Our research is directed toward elucidation of basic mechanisms involved in the production of cellular damage during exposure to oxidative stress and the contributions of such damage to aging and disease. Our current research involves studies in the following areas of research:[unreadable] [unreadable] (a) Regulation of apoptosis. Caspases, a family of cysteine proteases, play an important role in many forms of cell death by apoptosis and in pro-inflammatory cytokine maturation. Apoptosis is a process that eliminates damaged cells from tissues to permit replacement by new cells and thereby maintain integrity of the tissues. In continuing studies, we used mouse fibroblast cells to examine the effect of serum starvation on caspase-12 gene expression. Cells were first incubated with or without serum for 24 h and caspase-12 mRNA was monitored using semi-quantitative transcriptase polymerase chain reaction (RT-PCR ). We found that serum starvation induced caspase-12 mRNA expression. [unreadable] [unreadable] (b) Examination of apoptosis in unicellular organisms. In earlier studies, we studied the effects of oxidative stress on protein oxidation in a yeast strain (Saccharomyces cerevisiae) that lacks the gene (YCA1) that encodes synthesis of metacaspase, a homolog of mammalian caspase. We showed that exposure of this strain to hydrogen peroxide led to the generation of more oxidatively modified proteins than were observed in a yeast strain that contained the metacaspase gene. Since the underlying mechanisms of transcription and its regulation are fundamentally conserved from yeast to man, we have now used proteomic, genomic, bioinformatic tools, two-dimensional differential gel electrophoresis, mass spectroscopy, and microarray technologies to identify oxidized and phosphorylated proteins in the YCA1-deleted strain. Preliminary results show that a great number of proteins are carbonylated; tyrosine residues of proteins are phosphorylated; and elongation factors, 40s and 60s ribosomal subunits, and a protein involved in cysteine biosynthesis, O-acetyl homoserine sulfhydrylase, were up-regulated in the YCA1-deficient strain compared to the wild type. The identification of carbonylated and phosphorylated protein derivatives by mass spectrometry is in progress.[unreadable] [unreadable] (c) Identification of enzymes and regulatory proteins that are oxidized during starvation. In continuing studies carried in collaboration with members of the Proteomics Core Facility, we identified the enzymes and regulatory proteins that are oxidized to carbonyl derivatives during starvation for either nitrogen, carbon, or phosphate. Taking advantage of sophisticated proteomic analytical procedures, over 30 proteins were shown to be oxidized. These included phosphoserine aminotransferase, fructose-bisphosphate aldolase, acetylornithine deacetylase, ketol acid reductoisomerase, glutamic dehydrogenase, glutamine synthetase, aspartate aminotransferase, isomeroreductase, pyruvate dehydrogenase isocitrate dehydrogenase, succinyl-CoA synthetase beta chain, elongation-factor G, and elongation-factor TU.[unreadable] [unreadable] (d) Metal-catalyzed oxidation of mRNA leads to formation of dysfunctional polypetides. To investigate the effects of mRNA oxidation on translation ability, in vitro and in vivo metal-catalyzed oxidized or non-noxidized preparations of purified luciferase mRNA were subjected to translation in rabbit reticulocyte lysate or were transfected into HEK293 cells and incubated in the presence of paraquat. It was found that: (1) oxidation of the mRNA by either procedure led to the generation of 8-hydroxy-guanosine and other oxidized derivatives. (2) Translation of oxidized mRNA led to formation of short dysfunctional polypeptides, likely due to premature termination of translation events. (3) Although incapable of generating mature protein, the oxidized full-length mRNA did associate with ribosomes to form polysome mRNA complexes analogous to those obtained with non-oxidized mRNA. In contrast, similar studies in which oxidized mRNA encoding the cytoplasmic membrane protein, rhodopsin, was examined, short premature polypeptides, were not formed. Instead, high molecular weight species were formed. Furthermore, translation of this oxidized mRNA led to an increase in endoplasmic reticulum stress and an increase in caspase activity. Thus, our results indicate that mistranslation of mRNA oxidized in vivo or in vitro would have an impact on protein quality control, resulting in apoptosis as occurs in neurodegenerative diseases.[unreadable] [unreadable] (e) Denitration of nitrotyrosine. Nitric oxide is an important biological product that is used in a number of physiological processes. Unfortunately, over production of nitric oxide leads to a number of disorders. In particular, nitration of tyrosine residues of proteins inhibits their ability to be phosphorylated and thereby compromises important regulatory processes. The cyclic nitration and denitration of tyrosine residues could provide a means of maintaining reasonable levels of nitric oxide. Because of its potential importance, we have continued studies to determine the existence of mechanisms for denitration of tyrosine residues. To this end, we carried out studies with a tyrosine auxotrope yeast (Pichia pastoris) grown in media containing tyrosine, carbon, and nitrogen sources. Growth of this organism was greatly stimulated by further addition of nitrotyrosine. So far, we have determined that this strain uses nitrotyrosine for certain reactions that require tyrosine even though it is unable to use nitrotyrosine in the absence of tyrosine. Radioactively labeled nitrotyrosine will be used to confirm that the organism is using the nitrated compound. To increase our chances of success, we have obtained a strain of E. coli that requires tyrosine for growth. It will be grown under the same conditions as the yeast.[unreadable] [unreadable] (f) Role of oxidative stress in Progeria disease. Progeria is a genetic disease caused by mutation of the gene that produces lamin A, a structural protein that after its synthesis and post-translational modifications is located in the membrane of the cellular nucleus. The aim of this project is to determine if oxidative stress plays an important role in the aging process and subsequent death as measured by analysis of fibroblasts from these patients. We have grown fibroblasts of healthy controls, including the parents of the affected patient and a 10-year-old unaffected child, as well as ones from the patient. Our preliminary data shows that there is a lower ATP level in the patient?s fibroblasts than in the controls. Using the fluorescent probe, dichlorofluorescein diacetate (DCFH-DA), which is permeable to the cell membrane and reacts with ROS within the cell to yield highly fluorescent dichlorofluorescein (DCF), we observed a higher emission signal with the patient samples than with the healthy controls. This suggests that the levels of ROS are higher in the patient than in the controls. In further studies, the levels of protein carbonyls will be measured in samples at various passage numbers.